Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3.
Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating.
Bainbridge
G
G
Department of Biochemistry and Genetics, Medical School, University of Newcastle Upon Tyne, UK.
Mobasheri
H
H
Armstrong
G A
GA
Lea
E J
EJ
Lakey
J H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003553:Cystine; D004220:Disulfides; D004926:Escherichia coli / Q000502:physiology*; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D011994:Recombinant Proteins; D003553:Cystine